PROJECT SUMMARY Approximately one-third of postpartum cows develop uterine disease (metritis) within one week after calving and 5% of all calvings result in death of the cow within 30 days due to metritis or other complications that originate during calving. Postpartum uterine infection in women is less common, but approximately 10% of pregnancy-related deaths in the United States are caused by puerperal genital tract infection. Cows with early postpartum uterine disease will develop chronic inflammation of the uterus (endometritis or subclinical endometritis; SCE). Chronic endometritis in women (pelvic inflammatory disease; PID) is similar to the bovine disease and is caused by a variety of bacterial species. If left untreated, both SCE and PID cause infertility. The inability to effectively treat uterine disease and restore fertility may be partially explained by a critical gap in understanding of the uterine microbiome and its relationship to the disease status, uterine immune function, inflammation, tissue regeneration, and long-term programming of uterine dysfunction. The goal of this proposal is to understand the origins of uterine disease and the mechanisms through which uterine disease programs subfertility and infertility. The studies begin with the virgin bovine uterus where the presence and viability of the microbiome after first breeding will be determined (Aim 1; Study 1A & 1B). The microbiome of first and second pregnancy will then be studied (Aim 1, Study 1C). Whether metritis during the early postpartum period permanently damages the endometrium through structural and functional mechanisms and whether systemic antibiotic treatment mitigates the effect is the subject of Aim 2. Finally, the embryotrophic capacity of the diseased endometrium will be assessed (Aim 3). Collectively, the proposed investigations will elucidate novel structural and functional mechanisms through which the microbiome programs fertility. The research is innovative from both a conceptual and technical perspective. The uterine microbiome and its effect on fertility is an exciting new area of research and conceptually innovative. The cow provides an innovative model for uterine disease research due to the remarkable similarity between uterine disease states and outcomes in cows and women. Whole genome metagenomic sequencing will be performed as this approach will enable us to assess the entire microbiome. The transcriptome will be studied by using laser capture microdissection (LCM) and RNA sequencing. Reduced representation bisulfite sequencing (RRBS) will be used to identify patterns of DNA methylation associated with changes in the transcriptome. The proposed studies will identify tissue-specific mechanisms through which the endometrium fails to establish a receptive uterine environment and the cow fails to become pregnant. This fundamental knowledge is necessary to develop innovative tools to diagnose, treat, and ameliorate the effects of uterine disease. If disease mechanisms are understood then it may be possible to prevent tissue damage and (or) reverse the programming to fully restore fertility in women and or cattle that experience uterine disease.